This application is a 371 of PCT/FR93/01097, filed Nov. 08, 1993.
1. Field of the Invention
The present invention relates to novel polypeptides and to the genetic material which enables them to be expressed. More specifically, it relates to novel polypeptides having an opioid receptor activity.
2. Description of the Related Art
The opioid receptors have been known for a long time as membrane receptors of the nervous system, which receptors mediate the analgesic effects of alkaloid derivatives of opium. Endogenous ligands of these receptors and their precursors have been characterized, and their role in the response to pain and stress has been widely studied (Akil et al., (1984) Annu. Rev. Neurosci. 7, 223-255). Furthermore, pharmacological studies have revealed the existence of three subtypes of opioid receptors: mu (morphine), delta (enkephalin) and kappa (dynorphin) receptors. These same studies also demonstrated that the inhibitory action of the cellular activity of these receptors was linked to the activation of G proteins (Simonds, W. F. (1988) Endocrine Rev. 9, 200-212). For this reason, these receptors are nowadays classified within the family of receptors which interact with G proteins, that is a class of receptors possessing seven transmembrane domains and encompassing approximately 80% of known receptors.
Different laboratories have attempted to clone genes encoding opioid receptors. In particular, a protein which binds opioids with a selectivity of the mu type was purified from ox brain and partially sequenced. A cDNA was then isolated using nucleotide probes derived from this partial sequence. However, the protein which is deduced from this sequence does not possess any transmembrane domain and exhibits a high degree of homology with NCAM, an adhesion molecule (Schofield et al., (1989) EMBO J. 8, 489-495). More recently, the isolation of another cDNA has been described (Xie et al., (1992) Proc. Natl. Acad. Sci. USA 89, 4124-4128), which was obtained by expression cloning. The cDNA library was constructed from human placenta, which only expressed the kappa subtype, and it was screened using a peptide derivative of dynorphin by means of an affinity enrichment technique. However, this protein possesses a relatively weak affinity for opioid ligands, does not exhibit any subtype specificity, and, finally, seems very similar to the receptor for neuromedin K. Finally, all the attempts to clone opioid receptors by PCR, which are based on homologies with receptors coupled to G proteins, have been fruitless
The present invention describes, for the first time, the cloning of genes encoding opioid receptors. The present invention also describes, for the first time, the sequence of opioid receptors and their expression in recombinant cells. The present invention thus renders it possible to obtain an improved understanding of the structure of opioid receptors and to study their mechanism of action in more detail. The present invention also renders it possible to obtain opioid receptors of very high purity and in elevated quantity, thereby permitting functional and pharmacological studies to be carried out, antibodies to be made, etc. The invention also renders it possible to prepare opioid receptor fragments of defined size, as well as all types of opioid receptor derivatives. The invention also supplies recombinant cells which express opioid receptors or opioid receptor fragments which can be used for screening ligands of these receptors (agonists, antagonists, modulators, etc.). The DNA sequences of the invention also render it possible to make probes which are capable of detecting, in biological samples, any irregularity in the expression of an opioid receptor (non-expression, mutation, polymorphism, etc.). These probes can also be used for hybridization cloning of any other cDNA encoding an opioid receptor using tissues of various origins and, in particular, of human origin, as indicated further below.
An initial subject of the invention is, therefore, a nucleotide sequence which encodes a polypeptide having an opioid receptor activity. Within the meaning of the invention, opioid receptor comprises, in particular, the delta, mu and kappa subtypes.
Preferably, the invention relates to a nucleotide sequence which encodes a polypeptide having a delta opioid receptor activity.
Still more preferably, the nucleotide sequence according to the invention is selected from among:
(a) all or part of the nucleotide sequence SEQ ID NO: 1, or of its complementary strand,
(b) any sequence which hybridizes to a sequence (a) and which encodes a polypeptide having an opioid receptor activity, and
(c) the sequences derived from sequences (a) and (b) on account of the degeneracy of the genetic code.
A quite specific embodiment of the invention is represented by a nucleotide sequence which comprises all or part of the nucleotide sequence SEQ ID NO: 1, or of its complementary strand.
The various nucleotide sequences of the invention may or may not be of artificial origin. They can be genomic, cDNA or RNA sequences, hybrid sequences, or synthetic or semi-synthetic sequences. These sequences can be obtained, for example, by screening DNA libraries (cDNA library or genomic DNA library) with probes which are developed on the basis of the sequence SEQ ID NO: 1. Such libraries can be prepared from cells of different origins using standard molecular biological techniques which are known to the person skilled in the art. The nucleotide sequences of the invention can also be prepared by chemical synthesis, in particular using the phosphoramidite method, or else using mixed methods including chemical or enzymic modification of sequences which are obtained by screening libraries.
The nucleotide sequences of the invention may be used for producing opioid polypeptides. The term opioid polypeptide denotes any polypeptide having an opioid receptor activity, and any fragment or derivative of such a polypeptide. In order to produce opioid polypeptides, that part encoding the said polypeptide is generally placed under the control of signals which enable it to be expressed in a host cell. The choice of these signals (promoters, terminators, etc.) can vary depending on the host cell employed. To this end, the nucleotide sequences of the invention can be part of a vector, which can be autonomously replicating or integrating. More specifically, autonomously replicating vectors can be prepared using sequences which ensure autonomous replication in the chosen host. As regards integrating vectors, these can be prepared, for example, using sequences which are homologous with certain regions of the genome of the host, thereby allowing integration of the vector to take place by homologous recombination. The host cells which can be used for producing opioid polypeptides of the invention by the recombinant route are eukaryotic hosts as well as prokaryotic hosts. The suitable eukaryotic hosts which may be cited are animal cells, yeasts or fungi. In particular, as regards yeasts, those of the genera Saccharomyces, Kluyveromyces, Pichia, Schwanniomyces or Hansenula may be cited. As regards animal cells, those which may be cited are COS, CHO, C127 and NIH-3T3 cells, etc. Those fungi which may be cited more specifically are Aspergillus ssp. or Trichoderma ssp. Preference is given to employing the following bacteria E. coli, Bacillus or Streptomyces as prokaryotic hosts.
The nucleotide sequences of the present invention can also be used in the pharmaceutical sphere, either for preparing antisense sequences which can be used within the scope of gene therapy, or, once again, for preparing probes which render it possible to detect, by hybridization experiments, expression of opioid receptors in biological samples, and to demonstrate genetic anomalies (polymorphism or mutations), or aberrant expressions.
Inhibition of the expression of certain genes by antisense sequences has turned out to be a promising strategy for controlling gene activity. Antisense sequences are sequences whose transcription product is complementary to the coding strand of a given gene and is, for this reason, capable of specifically hybridizing to the transcribed mRNA, thereby inhibiting its translation into protein. Consequently, the invention relates to antisense sequences which are capable of at least partially inhibiting the production of opioid polypeptides as previously defined. Such sequences can consist of all or part of the nucleotide sequences defined above. Generally, they are sequences, or fragments of sequences, which are complementary to sequences encoding the peptides of the invention. Such sequences can be obtained from the sequence SEQ ID NO: 1, by means of fragmentation, etc., or by means of chemical synthesis.
As indicated above, the invention also permits synthetic or non-synthetic nucleotide probes to be prepared which are capable of hybridizing to the nucleotide sequences defined above which encode opioid polypeptides of the invention, or to the corresponding mRNAs. Such probes can be employed In vitro as a diagnostic tool for detecting expression of an opioid receptor, or else for detecting gene anomalies (faulty splicing, polymorphism, point mutations, etc.). In view of the numerous activities of the endogenous ligands of opioid receptors, the probes of the invention can thus render it possible to identify neurological, cardiovascular or psychiatric affections. These probes can also be used for detecting homologous nucleic acid sequences, which encode opioid polypeptides as previously defined, in, and isolating them from, other cellular sources, preferably cells of human origin. Although the present application is more specifically illustrated by a receptor of the delta type, biochemical and immunological studies described in the literature clearly indicate that the opioid receptors possess a significant degree of homology (same molecular weights, crossreactivity with the same antibodies, etc.). The probes of the invention generally comprise at least 10 bases, and they can comprise up to the whole of the SEQ ID NO: 1 sequence, or of its complementary strand. Preferably, these probes are labelled prior to being used. For this purpose, use can be made of various techniques known to the person skilled in the art (radioactive labelling, enzymic labelling, etc.). The hybridization conditions under which these probes can be used are indicated in the general cloning techniques below as well as in the examples.
The invention also relates to any polypeptide which results from the expression of a nucleotide sequence as previously defined. Preferably, the polypeptide is one which comprises all or part of the peptide sequence SEQ ID NO: 2, or of a derivative thereof.
Within the meaning of the present invention, the term derivative denotes any molecule which is obtained by genetic and/or chemical modification of the peptide sequence SEQ ID NO: 2. Genetic and/or chemical modification is to be understood to mean any mutation, substitution, deletion, addition and/or modification of one or more residues. Such derivatives can be generated for different purposes, such as, in particular, that of increasing the affinity of the peptide for its ligand(s), that of improving its levels of production, that of increasing its resistance to proteases, that of increasing and/or modifying its activity, or that of conferring on it novel pharmacokinetic and/or biological properties. Examples of derivatives which result from an addition and which may be cited are chimeric polypeptides which comprise an additional heterologous part which is linked to one end. The term derivative also encompasses polypeptides which are homologous with the SEQ ID NO: 2 polypeptide, which are derived from other cellular sources, in particular cells of human origin, or from other organisms, and which possess an activity of the same type. Such homologous polypeptides may be obtained by means of hybridization experiments as described in the examples.
Preferably, the polypeptides of the invention are polypeptides which possess the ability to bind morphine (mu type receptor), enkephalin (delta type receptor) or dynorphin (kappa type receptors). Still more preferably, the polypeptides are those which possess the ability to bind enkephalin (delta type receptor). Still in accordance with a preferred embodiment, the polypeptides of the invention are capable of being recognized by antibodies which recognize the complete SEQ ID NO: 2 peptide sequence. Antibodies of this nature can be generated by any technique known to the person skilled in the art, using the polypeptides described in the present application as antigens.
As indicated in the examples, these polypeptides can be expressed in different cell types in order to form functional opioid receptors.
The polypeptides of the invention can be obtained by the expression of a nucleotide sequence as described above in a host cell, by chemical synthesis on the basis of the SEQ ID NO: 2 sequence, using the techniques known to the person skilled in the art, or by a combination of these techniques.
The invention also relates to recombinant cells which are able to express, at their surface, a polypeptide having an opioid receptor activity. These cells may be obtained by introducing a nucleotide sequence as defined above and then culturing the said cells under conditions for expressing the said sequence.
The recombinant cells according to the invention can be either eukaryotic cells or prokaryotic cells. Suitable eukaryotic cells which may be cited are animal cells, yeasts or fungi. Those yeasts which may, in particular, be cited are yeasts of the genera Saccharomyces, Kluyveromyces, Pichia, Schwanniomyces or Hansenula. Those animal cells which may be cited are COS, CHO, C127 and NIH-3T3 cells, etc. Those fungi which may more specifically be cited are Aspergillus sup, or Trichoderma ssp. Preference is given to employing the following bacteria as prokaryotic cells: E. coli, Bacillus or Streptomyces. The cells which are thus obtained may be employed for measuring the ability of different molecules to behave as ligands or as modulators of the activity of the opioid receptors. More specifically, they may be employed in this way in a procedure for detecting and isolating ligands or modulators of opioid receptor activity, and, more preferably, agonists and antagonists.
The invention also relates, therefore, to a procedure for detecting and/or isolating ligands of the opioid receptors, according to which the following steps are carried out:
a molecule, or a mixture containing different molecules, which may not have been identified, is brought into contact with a recombinant cell as described above, which cell expresses, at its surface, a polypeptide having an opioid receptor activity, under conditions which permit interaction between the said polypeptide and the said molecule, if the latter possesses an affinity for the said polypeptide, and
the molecules bound to the said polypeptide are detected and/or isolated.
In a specific embodiment, this procedure of the invention is adapted for detecting and/or isolating agonists and antagonists of enkephalin for the delta opioid receptors.
The invention also relates to a procedure for detecting and/or isolating modulators of the opioid receptors, according to which the following steps are carried out:
a molecule, or a mixture containing different molecules, which may not have been identified, is brought into contact with a recombinant cell as described above, which cell expresses, at its surface, a polypeptide having an opioid receptor activity, in the presence of the endogenous ligand of the said receptor, under conditions which permit interaction between the said polypeptide and its ligand, and
the molecules which are capable of modulating the activity of the ligand on the said polypeptide are detected and/or isolated.
In a specific embodiment, this procedure of the invention is adapted for detecting and/or isolating modulators of the activity of enkephalin on the delta opioid receptors.
The invention also relates to the use of a ligand or of a modulator which is identified and/or obtained according to the procedure described above as a medicament. This is because such ligands or modulators can render it possible to treat certain affections which are linked to the opioid receptors. Specifically, since the opioid receptors mediate an analgesic effect, agonists of these receptors can be used to diminish pain sensations. Furthermore, since these receptors are mediators of the effects of opium derivatives, their antagonists can be employed within a disintoxication treatment.
The invention also relates to any medicament which includes, as active principle, at least one molecule which acts on a receptor of the invention. Preferably, the molecule is a ligand or a modulator which is identified and/or isolated according to the previously described procedure.